Antibodies recognize specific antigens and thereby provoke various in vivo phenomena. Thus, antibodies play an important role in in vivo defense mechanisms. In particular, since antibodies have antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), which are effective for elimination of cancer cells and the like, they have been used as anticancer agents. There are many antibody agents, which have been produced as a result of focusing the above-mentioned activities and have been put into practical use. Among such antibody agents, a considerable number of agents exhibit good therapeutic effects. In addition to the intended use as pharmaceutical agents, antibodies have been widely used as, for example, various diagnostic agents, or useful tools for research and development. Accordingly, it is desired to produce an antibody which recognizes, as an antigen, a protein that plays an important role in various in vivo phenomena.
An example of such a protein that plays an important role in in vivo phenomena is a protein that penetrates to a cell membrane (hereinafter referred to as a “transmembrane protein”). Examples of such a transmembrane protein include various types of receptor proteins and ion channel proteins. A majority of such transmembrane proteins are associated with transmission and/or migration of information and substances inside and outside a cell, and thereby they play an important role in the survival, growth, differentiation and the like of the cell. Thus, it is extremely significant to produce an antibody capable of recognizing the above-described transmembrane protein, which can be used as the aforementioned pharmaceutical agent, diagnostic agent, or research and development tool.
To date, there have been developed various methods for producing an antibody directed against any given antigen (a hybridoma production method, a DNA immunization method, a phage display method, etc.). Particularly, in recent years, a method, referred to as an ADLib system (or an ADLib method), has attracted considerable attention as a technique of easily producing a large amount of antibody having high specificity (see Patent Literature 1 and Non Patent Literature 1). According to this method, an antibody having desired specificity and affinity can be provided by a simple method.
However, the production of an antibody directed against a transmembrane protein according to the above-described various methods for producing antibodies has been problematic in the following respects.
In the method of directly administering a transmembrane protein-expressing cell as an antigen to an animal to be immunized, so as to induce an immune response, the transmembrane protein is expressed in a physiologically three-dimensional structure on the protein-expressing cell membrane. Since the protein-expressing cell undergoes protein degradation in vivo, it is difficult to produce an antibody directed against the transmembrane protein that is in a physiological state.
The DNA immunization method comprises incorporating the cDNA of a transmembrane protein into a suitable mammalian cell expression vector and then directly administering the expression vector to an animal to be immunized (see Non Patent Literature 2). According to this method, the three-dimensional structure of the transmembrane protein is likely to be reproduced on a cell membrane in vivo. However, the action mechanism of the protein is still unknown, and thus, whether or not an antibody can be produced directed against the transmembrane protein that is in a physiological state has not yet been clarified.
The phage display method is a system whereby an antibody variable region gene is fused with a coat protein of a filamentous phage as one type of Escherichia coli virus, such that the infectious ability of the phage is not lost, and the gene is allowed to express as a fusion protein (see Non Patent Literatures 3 and 4). In this method, when phage particles are allowed to react with a transmembrane protein and a desired antibody is selected, the use of a purified transmembrane protein is required, and whether or not the transmembrane protein as a target maintains physiological functions is not guaranteed.
The ADLib method, which promotes somatic homologous recombination that occurs in immunocytes so that a variety of antibody molecules are presented on the surfaces of the immunocytes, also has the above-described problems. Hence, it is not always easy to select an antibody directed against a transmembrane protein which is hardly purified in a physiological state.
Given the above, it has been difficult for all of the production methods to produce an antibody directed against a transmembrane protein.